Mutant p53 protects triple-negative breast adenocarcinomas from ferroptosis in vivo

Abstract

The TP53 tumor suppressor gene is mutated early in most of the patients with triple-negative breast cancer (TNBC). The most frequent TP53 alterations are missense mutations that contribute to tumor aggressiveness. Here, we used an autochthonous somatic TNBC mouse model, in which mutant p53 can be toggled on and off genetically while leaving the tumor microenvironment intact and wild-type for p53 to identify physiological dependencies on mutant p53. In TNBCs that develop in this model, deletion of two different hotspot p53R172H and p53R245W mutants triggers ferroptosis in vivo, a cell death mechanism involving iron-dependent lipid peroxidation. Mutant p53 protects cells from ferroptosis inducers, and ferroptosis inhibitors reverse the effects of mutant p53 loss in vivo. Single-cell transcriptomic data revealed that mutant p53 protects cells from undergoing ferroptosis through NRF2-dependent regulation of Mgst3 and Prdx6, which encode two glutathione-dependent peroxidases that detoxify lipid peroxides. Thus, mutant p53 protects TNBCs from ferroptotic death.

Fig. 1.. Mutant p53 protects breast adenocarcinomas from ferroptosis in vivo.

(A) Representative hematoxylin and eosin (H&E)– and 4HNE-stained sections of P¹⁷²CC adenocarcinomas treated with AAV-Control (left) or AAV-Δmut-p53 (right) and quantification of lipid droplets in three fields of view per sample from AAV-Control (n = 3) 5, and 12 days after last AAV injection or AAV-Δmut-p53 (n = 3) 2, 4, and 8 days after last AAV injection. Yellow outline, lipid droplets. H&E shown is from a tumor harvested on day 8 after last AAV injection. Image for the 4HNE is from a tumor harvested on day 2 after last AAV injection. Scale bars, 500 μm. (B) Red Oil O staining of a tumor treated with AAV-Control and AAV-Δmut-p53 (P¹⁷²CC mice). Image is from tumor harvested on day 2 after last AAV injection. (C) Representative H&E- and 4HNE-stained sections of P²⁴⁵CC adenocarcinomas treated with AAV-Control (left) or AAV-Δmut-p53 (right) and quantification of lipid droplets within the tumor section in three fields of view per sample from AAV-Control (n = 5) or AAV-Δmut-p53 (n = 8) at 3 days after second treatment. L, lumen of malignant epithelium; yellow outline, lipid droplets. Scale bars, 500 μm. (D) Red Oil O staining of a tumor treated with AAV-Δmut-p53 (P²⁴⁵CC mice). (E) Gene set enrichment analysis (GSEA) of glutathione metabolic processes and oxidoreductase activity on Ch_CH donors and NAD_NADP acceptors in breast cancer patients with TP53 missense mutations (n = 214) as compared with patients with TP53 (n = 110) truncating mutations. (F) Tumor growth rates (mm³/day) at day 3 (left) or end point (right) for adenocarcinomas from P¹⁷²CC (n = 12) and P²⁴⁵CC (n = 6) adenocarcinomas treated with AAV-Control (n = 5), AAV-Δmut-p53 (n = 10), or AAV-Δmut-p53 plus liproxstatin-1 (n = 3). Data are means ± SEM. Significant differences were evaluated by Student’s t test [(A) and (C)] and one-way analysis of variance (ANOVA) (F). *P < 0.05 and **P < 0.01. TCGA, The Cancer Genome Atlas. RNA-seq, RNA sequencing; NES, normalized enrichment score; FDR, false discovery rate.

Fig. 2.. Mutant p53 protects breast adenocarcinomas from ferroptosis through GPX4.

(A) Viability of P¹⁷²CC (610) and its corresponding p53-deleted clone (P^ΔCC) treated with ferroptosis inducers RSL3, ML162, ML210, and ferroptosis inhibitor liproxstatin-1 at the indicated concentrations. Liproxstatin-1 was added 30 min before RSL3, ML162, or ML210. Viability was assessed 24 hours after treatment. Dotted line denotes viability of cells treated with sham dimethyl sulfoxide (DMSO). (B) Viability of P²⁴⁵CC (1128) and its corresponding p53-deleted clone (P^ΔCC) treated with RSL3, ML162, ML210, and liproxstatin-1 at the indicated concentrations. Liproxstatin-1 was added 30 min before treatment with RSL3, ML162, or ML210. Viability was assessed 24 hours after treatment. Dotted line denotes viability of cells treated with sham DMSO. (C) Viability of P¹⁷²CC (left) and P²⁴⁵CC (right) cell lines and their corresponding p53-knockout clones (P^ΔCC) treated with RSL3 or sham at high confluency (1 × 10⁴ cells per well). Viability was assessed 24 hours after treatment. (D) Viability of P¹⁷²CC (610, left) and p53R245W germline breast tumor cells (391, right) infected with a doxycycline-inducible lentivirus expressing sgRNAs targeting Trp53 and treated with sham or doxycycline (1 μg/ml) for 48 hours and subsequently treated with RSL3 for 24 hours. Significant differences between groups were evaluated by Student’s t test for (A) to (D) *P < 0.5, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n.s., not significant.

Fig. 3.. scRNA-seq analysis of AAV-Δmut-p53 treated TNBCs.

(A) Top: Treatment regimen; arrows denote the timing of AAV injections. Bottom: Waterfall plot of percent changes in volumes of P²⁴⁵CC adenocarcinomas treated with AAV-Control (n = 5) or AAV-Δmut-p53 (n = 8) at day 6. * denotes samples submitted for scRNA-seq, which contained lipid-like structures and/or were 4HNE positive. (B) UMAP of labeled cells pooled and separated by treatment: AAV-Control (n = 2) or AAV-Δmut-p53 (n = 5) at day 3 after second AAV treatment. (C) UMAP of cells expressing selected epithelial markers (Epcam, Krt8, Krt18, and Krt19) identified four epithelial clusters. (D) Proportions of tumor cells in each of the four clusters by treatment type. Each dot represents one spontaneous tumor. (E) Violin plots of Ftl1 and Fth1 from the scRNA-seq from epithelial clusters (1, 9, 11, 14) showing the expression levels of genes that are enriched in AAV-Δmut-p53–injected tumors when compared to AAV-Control tumors. ***P = 1.041 × 10⁻¹⁶⁶ (Fth1) and ***P = 1.2755 × 10⁻⁶⁰ (Ftl1). (F) Top 4 pathways identified by GSEA in each tumor cell cluster. (G) Heatmap of GSVA scores indicating differentially activated pathways in pooled tumor cells grouped by treatment type. * denotes immune-related pathways activated.

Fig. 4.. Mutant p53 mitigates oxidative stress and ferroptosis through regulation of two enzymes, Mgst3 and Prdx6.

(A) Violin plots of genes from the scRNA-seq associated with the ROS pathway from epithelial clusters (1, 9, 11, 14, from Fig. 3C) showing the expression levels of genes that are enriched in AAV-Control or AAV-Δmut-p53 injected tumors. (B and C) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of Mgst3 and Prdx6 in P¹⁷²CC (B) and P²⁴⁵CC (C) cells and their respective Trp53-deleted clones. (D) Flow cytometric analysis of p53R245W (391) cells with or without Mgst3 or Prdx6 silencing at 48 hours and stained with C11-BODIPY, a marker of lipid oxidation. Efficiency of mRNA-mediated silencing is shown in fig. S4A. (E and F) Viability of P¹⁷²CC (E) and P²⁴⁵CC (F) cells after siRNA silencing of Mgst3 or Prdx6 and treatment with DMSO or RSL3. Viability was assessed 24 hours after treatment. Efficiency of mRNA-mediated silencing shown in fig. S4B. (G) Viability of P^ΔCC (1128) cells transduced with lentiviral particles overexpressing (OE) cDNAs for GFP, Mgst3, or Prdx6 and treated with RSL3 (5 μM). Viability was assessed 24 hours after treatment. Efficiency of lentivirus-mediated overexpression of Mgst3 and Prdx6 is shown in S4C. (H) Levels or Mgst3 or Prdx6 expression in P²⁴⁵CC cells after short hairpin RNA targeting Mgst3 of Prdx6, respectively. (I) Tumor growth rates of these cells after injection into nude mice and treatment with RSL3 intratumorally at indicated time points (arrows). Data are means ± SEM. Significant differences between groups was evaluated by one-way ANOVA [(B), (D), and (E)] and Student’s t test [(C) and (G)] and two-way ANOVA with Dunnett’s multiple comparison (I). *P < 0.5, **P < 0.01, and ****P < 0.0001. GFP, green fluorescent protein.

Fig. 5.. Mutant p53 protects human TNBCs against ferroptosis.

(A) RT-qPCR data showing transcript levels of TP53, MGST3, and PRDX6 in listed breast tumor cell lines with or without siRNA silencing of mutant TP53. p53 mutations for each cell line are listed. (B) Viability of human patient breast cancer cells from (A) treated with RSL3. Viability was assessed 24 hours after treatment. (C) Tumor growth of MDA-MB-231 cells expressing short hairpin targeting MGST3 or PRDX6 in nude animals (N = 4 to 5 per group) injected with low (0.5 × 10⁶ at various time points, left) or high (35 × 10⁶ at 42 days, right) cell numbers. (D) Kaplan-Meier overall survival curves for breast cancer patients with tumors expressing high levels (80% threshold; 214 patients) or low levels (20% threshold; 214 patients) of our six-gene NRF2 dataset (ATOX1, GCLM, PRDX2, PRDX6, MGST3, and TXN) with a log-rank P value of 9.6 × 10⁻⁰⁵. These analyses were done using GEPIA2. Data are means ± SEM. Significant differences between groups was evaluated by Student’s t test for (A) and (B). *P < 0.5, **P < 0.01, and ***P < 0.001.

Fig. 6.. Mutant p53 inhibits ferroptosis through the NRF2-dependent regulation of Mgst3 and Prdx6.

(A) ENRICHR results for the top transcription factors of our mutant-p53/oxidative stress 6-gene dataset. (B) One hundred eighty DEGs regulated by mutant p53 in clusters 1, 9, 11, and 14 were examined for AREs. (C) Violin plots of seven DEGs from cancer clusters regulated by mutant p53 that contain AREs in their promoters. (D) RT-qPCR analysis for Prdx6 and Mgst3 in P²⁴⁵CC cells treated with brusatol for 3 hours. (E) Viability of P²⁴⁵CC cells with or without siRNA silencing of NRF2 and treated with RSL3 (10 μM) or DMSO. (F) Viability of P²⁴⁵CC cells treated with RSL3 (5 μM), brusatol (0.5 μM), or both (combo). (G) Cell viability of indicated cells treated with RSL3 (5 μM), brusatol (10 μM for MDA-MB-231 and 1.0 μM for HCC1395), or both. (H) Co-immunoprecipitation of mutant p53 and NRF2. IP, immunoprecipitation; IB, immunoblotting. (I) Purified NRF2-His bound p53R245W-Flag but not negative controls Pla2g16-Flag or p53N15fs-Flag (first 15 amino acids of p53, followed by a truncation and a frameshift). Membranes were probed with Flag and NRF2 antibodies. (J and K) CUT&RUN RT-qPCR for two ARE sequences within the Prdx6 promoter using anti-NRF2 or immunoglobulin G (IgG) (J) or anti-p53 antibodies in P²⁴⁵CC (K). Cells with CRISPR knockout of mutant p53 were used as a baseline (K). (L and M) Co-immunoprecipitation of mutant p53 and NRF2 from lysates (L) and viability (M) in P^−/−CC (1374) cells transduced with indicated lentiviruses. NRF2 pull-down was quantified as ratio of pull-down to input. (N) NFE2L2 (NRF2) dependency of human cancer cell lines with missense TP53 (n = 756) or wild type (n = 636). Data are means ± SEM. Significant differences were evaluated by Student’s t test [(E), (J), and (K)] one-way ANOVA [(D), (F), (G), and (M)]. **P < 0.01, ***P < 0.001, and ****P < 0.0001.